Remedies for myeloma to be used together with nitrogen mustard antitumor agents

ABSTRACT

A therapeutic agent for myeloma comprising a combined use of a nitrogen mustard anticancer agent and anti-IL-6 receptor antibody. Thus, a therapeutic agent for myeloma comprising anti-IL-6 receptor antibody for use in combination with a nitrogen mustard anticancer agent; a therapeutic agent for myeloma comprising a nitrogen mustard anticancer agent for use in combination with anti-IL-6 receptor antibody; and a therapeutic agent for myeloma comprising a nitrogen mustard anticancer agent and anti-IL-6 receptor antibody.

TECHNICAL FIELD

[0001] The present invention relates to a pharmaceutical composition forcombined use of nitrogen mustard anticancer agents with anti-IL-6receptor antibody for treatment of myeloma.

BACKGROUND ART

[0002] For chemotherapy of human tumors, alkylating agents,antimetabolites, antitumor antibiotics, platinum compounds and the likehave been used. When single uses of these activating agents do notexhibit marked therapeutic effects, therapies in which multiple drugsare used in combination have been considered (Frei, E. III, Cancer Res.(1992) 32, 2593-2607). As anticancer agents that belong to thealkylating agents, there are mentioned nitrogen mustard anticanceragents, which is a general term used for the anticancer agents that havea partial structure called nitrogen mustard. Of the nitrogen mustardanticancer agents melphalan has been put into practical use.

[0003] IL-6 is a multifunctional cytokeine called B-cell stimulatoryfactor 2 or interferon β2. IL-6 was discovered as a differentiationfactor responsible for activation of B-lymphocytes (Hirano, T. et al.,Nature (1986) 324, 73-76). Thereafter, it was found to be amultifunctional cytokeine that influences the function of various cells(Akira, S. et al., Adv. in Immunology (1993) 54, 1-78). IL-6 imparts itsbiological activity through two proteins on the cell membrane.

[0004] One of them is a ligand-biding protein with a molecular weight ofabout 80 kD, IL-6 receptor, to which IL-6 binds. IL-6 receptor occursnot only in a membrane-bound form that penetrates and is expressed onthe cell membrane but also as a soluble IL-6 receptor consisting mainlyof the extracellular region. The other is non-ligand-binding gp130 witha molecular weight of about 130 kD that takes part in signaltransmission. IL-6 and IL-6 receptor form a IL-6/IL-6 receptor complex,to which another membrane protein gp130 is bound, and thereby thebiological activity of IL-6 is transmitted to the cell (Taga et al., J.Exp. Med. (1987) 166, 967).

[0005] Antibodies to IL-6 receptor (anti-IL-6 receptor antibodies) havebeen known (Novick D. et al., Hybridoma (1991) 10, 137-146 Huang, Y. W.et al., Hybridoma (1993) 12, 621-630 International Patent ApplicationWO95-09873, French Patent Application FR 2694767, U.S. Pat. No.5,216,128), one of which is PM-1 derived from mice (Hirata et al., J.Immunology (1989) 143, 2900-2906). Furthermore, a reshaped antibodyobtained by replacing the complementarity determining regions (CDRs) ofthe mouse antibody with the CDRs of a human antibody has also beenknown.

[0006] However, the combined use of a nitrogen mustard anticancer agentand IL-6 receptor as a therapeutic agent for treatment of myeloma hasnot been known.

DISCLOSURE OF THE INVENTION

[0007] It is an object of the present invention to provide a new type ofmyeloma therapeutic agent that is more effective than the conventionallyknown myeloma therapeutic agents.

[0008] As a result of an intensive study to solve the above problems,the applicants have found that the combination of a nitrogen mustardanticancer agent, a conventionally known anticancer agent, and anti-IL-6receptor antibody has a synergistic effect, i.e. it is more effectivethan the sole use of the nitrogen mustard anticancer agent or the soleuse of anti-IL-6 receptor antibody for treatment of myeloma, and havecompleted the present invention.

[0009] Thus, the present invention provides a therapeutic agent fortreatment of myeloma comprising anti-IL-6 receptor antibody for use incombination with a nitrogen mustard anticancer agent.

[0010] The present invention also provides a therapeutic agent fortreatment of myeloma comprising anti-IL-6 receptor monoclonal antibodyfor use in combination with a nitrogen mustard anticancer agent.

[0011] The present invention also provides a therapeutic agent fortreatment of myeloma comprising PM-1 antibody for use in combinationwith a nitrogen mustard anticancer agent.

[0012] The present invention also provides a therapeutic agent fortreatment of myeloma comprising a reshaped PM-1 antibody for use incombination with a nitrogen mustard anticancer agent.

[0013] The present invention also provides a therapeutic agent fortreatment of myeloma comprising anti-IL-6 receptor antibody for use incombination with mechlorethamine, nitrogen mustard N-oxide, melphalan,uramustin, ifosfamide, chlorambucil, or cyclophosphamide.

[0014] The present invention also provides a therapeutic agent fortreatment of myeloma comprising a reshaped human PM-1 antibody for usein combination with melphalan.

[0015] The present invention also provides a therapeutic agent fortreatment of myeloma comprising a nitrogen mustard anticancer agent foruse in combination with anti-IL-6 receptor antibody.

[0016] The present invention also provides a therapeutic agent fortreatment of myeloma comprising a nitrogen mustard anticancer agent foruse in combination with anti-IL-6 receptor monoclonal antibody.

[0017] The present invention also provides a therapeutic agent fortreatment of myeloma comprising a nitrogen mustard anticancer agent foruse in combination with PM-1 antibody.

[0018] The present invention also provides a therapeutic agent fortreatment of myeloma comprising a nitrogen mustard anticancer agent foruse in combination with a reshaped human PM-1 antibody.

[0019] The present invention also provides a therapeutic agent fortreatment of myeloma comprising mechlorethamine, nitrogen mustardN-oxide, melphalan, uramustin, ifosfamide, chlorambucil, orcyclophosphamide in combination with anti-IL-6 receptor antibody.

[0020] The present invention also provides a therapeutic agent fortreatment of myeloma comprising melphalan for use in combination with areshaped human PM-1 antibody.

[0021] The present invention also provides a therapeutic agent fortreatment of myeloma comprising a nitrogen mustard anticancer agent andanti-IL-6 receptor antibody.

[0022] The present invention also provides a therapeutic agent fortreatment of myeloma comprising a nitrogen mustard anticancer agent andanti-IL-6 receptor monoclonal antibody.

[0023] The present invention also provides a therapeutic agent fortreatment of myeloma comprising a nitrogen mustard anticancer agent andPM-1 antibody.

[0024] The present invention also provides a therapeutic agent fortreatment of myeloma comprising a nitrogen mustard anticancer agent anda reshaped human PM-1 antibody.

[0025] The present invention also provides a therapeutic agent fortreatment of myeloma comprising mechlorethamine, nitrogen mustardN-oxide, melphalan, uramustin, ifosfamide, chlorambucil, orcyclophosphamide and anti-IL-6 receptor antibody.

[0026] The present invention provides a therapeutic agent for treatmentof myeloma comprising melphalan and a reshaped human PM-1 antibody.

BRIEF EXPLANATION OF DRAWINGS

[0027]FIG. 1 is a graph showing the relationship of anti-human IL-6receptor antibody concentration and melphalan concentration with thegrowth (incorporation of ³H-labeled thymidine) of a human myeloma cellline in the presence of 0.1 ng/ml IL-6.

[0028]FIG. 2 is a graph showing the relationship of anti-human IL-6receptor antibody concentration and melphalan concentration with thegrowth (incorporation of ³H-labeled thymidine) of a human myeloma cellline in the presence of 1 ng/ml IL-6.

[0029]FIG. 3 is a graph showing the relationship of anti-human IL-6receptor antibody concentration and adriamycin concentration with thegrowth (incorporation of ³H-labeled thymidine) of a human myeloma cellline in the presence of 0.1 ng/ml IL-6.

[0030]FIG. 4 is a graph showing the relationship of anti-human IL-6receptor antibody concentration and adriamycin concentration with thegrowth (incorporation of ³H-labeled thymidine) of a human myeloma cellline in the presence of 1 ng/ml IL-6.

[0031]FIG. 5 is a graph showing the relationship of anti-human IL-6receptor antibody concentration and vincristine concentration with thegrowth (incorporation of ³H-labeled thymidine) of a human myeloma cellline in the presence of 0.1 ng/ml IL-6.

[0032]FIG. 6 is a graph showing the relationship of anti-human IL-6receptor antibody concentration and vincristine concentration with thegrowth (incorporation of ³H-labeled thymidine) of a human myeloma cellline in the presence of 1 ng/ml IL-6.

[0033]FIG. 7 is a graph showing the survival days of the mice implantedwith human myeloma cells in a single-drug administration (1 mg/kg) ofanti-human IL-6 receptor antibody (hPM-1) and melphalan, or in acombined use thereof.

[0034]FIG. 8 is a graph showing the amount of M protein in the miceimplanted with human myeloma cells in a single-drug administration (1mg/kg) of anti-human IL-6 receptor antibody (hPM-1) and melphalan, or ina combined use thereof.

[0035]FIG. 9 is a graph showing the survival days of mice implanted withhuman myeloma cells in a single-drug administration (3 mg/kg) ofanti-human IL-6 receptor antibody (hPM-1) and-melphalan, or in acombined use thereof, with a synergistic effect obtained by the combineduse.

[0036]FIG. 10 is a graph showing the changes in body weight of miceimplanted with human myeloma cells in a single-drug administration ofanti-human IL-6 receptor antibody (hPM-1) and melphalan, or in acombined use thereof.

[0037]FIG. 11 is a graph showing the amount of M protein in the serum ofmice implanted with human myeloma cells at 30 days after the tumorimplantation in the anti-human IL-6 receptor antibody (hPM1) single-drugadministration group and in the melphalan single-drug administrationgroup.

[0038]FIG. 12 is a graph showing the amount of M protein in the serum ofmice implanted with human myeloma cells at 35 days after the tumorimplantation in the melphalan single-drug administration group and theanti-human IL-6 receptor antibody (hPM1)-combined administration group.

[0039]FIG. 13 is a graph showing the amount of M protein in the serum ofmice implanted with human myeloma cells at 42 days after the tumorimplantation in the melphalan single-drug administration group and theanti-human IL-6 receptor antibody (hPM1)-combined administration group.

[0040]FIG. 14 is a graph showing the survival period by a survival curveof mice implanted with human myeloma cells in the melphalan single-drugadministration group and the anti-human IL-6 receptor antibody(hPM1)-combined administration group, indicating an enhanced effect bycombined use.

[0041]FIG. 15 is a graph showing the changes in body weight of miceimplanted with human myeloma cells in the anti-human IL-6 receptorantibody single-drug administration group.

[0042]FIG. 16 is a graph showing the changes in body weight of miceimplanted with human myeloma cells in the melphalan single-drugadministration group and the anti-human IL-6 receptor antibody(hPM1)-combined administration group.

EMBODIMENTS FOR CARRYING OUT THE INVENTION

[0043] Nitrogen mustard anticancer agents for use in the presentinvention is a general term for the anticancer agents having a partialstructure called nitrogen mustard having the structure:

[0044] examples of which agents include:

[0045] Mechlorethamine,

[0046] Nitrogen mustard N-oxide (methyl-bis(β-chloroethyl) amine N-oxidehydrochloride),

[0047] Melphalan (p-[bis(2-chloroethyl)amino-L-phenylalanine),

[0048] Chlorambucil (p-bis(2-chloroethyl)amino-phenylbutyric acid),

[0049] Uramustin (5-bis(2-chloroethyl)aminouracil),

[0050] Ifosmide(N,N′-bis(2-chloroethyl)-N′, O-propylenephosphoric acidester diamide

[0051] Cyclophosphamide(N,N′-bis(β-chloroethyl)-N′,O-propylenephosphoric acid ester diamide,

[0052] and the like.

[0053] In accordance with the present invention, these nitrogen mustardanticancer agents may be used as a single drug or in combination. Amongthem, melphalan is also called sarcolysine or L-phenylalanine mustardand has the following structure:

[0054] Mechlorethamine can be obtained by a known method, for example, amethod described in Abrams et al., J. Soc. Chem. Ind. (London) (1949)68, 280.

[0055] Nitrogen mustard N-oxide can be obtained by a known method, forexample, a method described in Aiko et al., J. Pharm. Soc. Japan (1952)72, 1297.

[0056] Melphalan can be obtained by a known method, for example, amethod described in Bergel, F. et al., J. Chem. Soc. (1954) 2409.

[0057] Chlorambucil can be obtained by a known method, for example, amethod described in Balazc, M. K. et al., J. Pharm. Sci. (1970) 59, 563.

[0058] Uramustin can be obtained by a known method, for example, amethod described in Lyttle and Petering, J. Am. Chem. Soc. (1958) 80,6459.

[0059] Ifosfamide can be obtained by a known method, for example, amethod described in Arnold H. et al., U.S. Pat. No. 3,732,340, (1973 toAsta) or Brassfield, HJ. A. et al., J. Am. Che. Soc. (1975) 97, 4143.

[0060] Cyclophosphamide can be obtained by a known method, for example,a method described in Arnold H. et al., Angew. Chem. (1958) 70, 539.

[0061] 1. Anti-IL-6 Receptor Antibody

[0062] Anti-IL-6 receptor antibodies for use in the present inventionmay be of any origin, any kind (monoclonal or polyclonal), and any form,as long as they have a higher therapeutic effect for myeloma whenadministered in combination with a nitrogen mustard anticancer agentthan when an anti-IL-6 receptor antibody alone is administered or when anitrogen mustard anticancer agent alone is administered.

[0063] Anti-IL-6 receptor antibodies for use in the present inventioncan be obtained as polyclonal or monoclonal antibodies using a knownmethod. As the anti-IL-6 receptor antibodies for use in the presentinvention, monoclonal antibodies of, in particular, a mammalian origin,are preferred. Monoclonal antibodies of a mammalian origin include thoseproduced by a hybridoma and recombinant antibody produced by a hostwhich has been transformed with an expression vector containinggenetically engineered antibody genes. Anti-IL-6 receptor antibodies foruse in the present invention, via binding to IL-6 receptor, block thebinding of IL-6 to IL-6 receptor, and thereby inhibit signaltransmission of IL-6, and therefore are antibodies which inhibit thebiological activity of IL-6.

[0064] Examples of such antibodies include PM-1 antibody (Hirata, etal., J. Immunology (1989) 143, 2900-2906), or AUK12-20 antibody, AUK64-7antibody or AUK146-15 antibody (International Patent Application WO92-19759), and the like. Of them, PM-1 antibody is most preferred.

[0065] Incidentally, the hybridoma cell line which produces PM-1antibody has been internationally deposited under the provisions of theBudapest Treaty as PM-1 on Jul. 10, 1990 with the National Institute ofBioscience and Human Technology, Agency of Industrial Science andTechnology, of 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki, Japan, asFERM BP-2998.

[0066] 2. Antibody Produced by Hybridoma

[0067] Monoclonal antibodies can be obtained by constructing a hybridomausing basically a known procedure as described bellow. Thus, IL-6receptor is used as an immunizing antigen and is immunized in theconventional method of immunization. The immune cells thus obtained arefused with known parent cells in the conventional cell fusion process,and then screened by the conventional screening method to screenmonoclonal antibody-producing cells.

[0068] Specifically, monoclonal antibodies may be obtained in thefollowing manner.

[0069] For example, IL-6 receptor used as the immunizing antigen forobtaining antibody is not limited to any animal species, but IL-6receptor derived from humans is particularly preferred. For human IL-6receptor, IL-6 receptor protein can be obtained using a gene sequencedisclosed in European Patent Application EP 325474. There are two kindsof IL-6 receptor: IL-6 receptor expressed on the cell membrane, and IL-6receptor detached from the cell membrane (Soluble IL-6 Receptor;Yasukawa et al., J. Biochem. (1990) 108, 673-676).

[0070] Soluble IL-6 receptor is composed mainly of the extracellularregion of IL-6 receptor bound to the cell membrane, and soluble IL-6receptor is different from the membrane-bound IL-6 receptor in that theformer lacks the transmembrane region or both of the transmembraneregion and the intracellular region. In accordance with the presentinvention, IL-6 receptor used as the immunizing antigen may be eitherthe membrane-bound or the soluble IL-6 receptor. Alternatively, it maybe a mutant thereof.

[0071] After a gene encoding IL-6 receptor is inserted into a knownexpression vector to transform an appropriate host cell, the desiredIL-6 receptor protein is purified from the host cell or a culturesupernatant thereof using a known method, and the IL-6 receptor proteinthus purified may be used as the immunization antigen. Alternatively,cells that express IL-6 receptor protein may be used as the immunizationantigen.

[0072] Preferably mammals to be immunized with the immunization antigenare selected in consideration of their compatibility with the parentcells for use in cell fusion and they generally include, but are notlimited to, rodents, logomorphas, and primates.

[0073] As rodents, for example, mice, rats, hamsters, etc. are used. Aslogomorphas, for example, rabbits are used. As primates, for example,monkeys are used. As monkeys, catarrhines (Old-World monkeys) such ascynomolgi (crab-eating macaque), rhesus monkeys, sacred baboons,chimpanzees etc. are used.

[0074] Immunization of animals with an immunization antigen is carriedout using a known method. A general method, for example, involvesintraperitoneal or subcutaneous administration of an immunizationantigen to the mammal. Specifically, an immunization antigen which wasdiluted and suspended in an appropriate amount of phosphate bufferedsaline (PBS) or physiological saline etc. is mixed with an appropriateamount of Freund's complete adjuvant. After being emulsified, it ispreferably administered to a mammal for several times every 4 to 21days. Additionally a suitable carrier may be used at the time ofimmunization of the immunization antigen.

[0075] After the immunization and confirmation of the increase in thedesired antibody levels in the serum by a conventional method, immunecells are taken out from the mammal and are subjected to cell fusion, inwhich especially preferred immune cells are the spleen cells.

[0076] The mammalian myeloma cells as the other parent cells which aresubjected to cell fusion with the above-mentioned immune cellspreferably include various known cell lines such as P3 (P3×63Ag8.653)(Kearney, J. F. et al., J. Immunol. (1979) 123, 1548-1550), P3×63Ag8U·U1(Yelton, D. E. et al., Current Topics in Microbiology and Immunology(1978) 81, 1-7), NS-1 (Kohler, G. and Milstein, C., Eur. J. Immunol.(1976) 6, 511-519), MPC-11 (Margulies, D. H. et al., Cell (1976) 8,405-415), SP2/0 (Shulman, M. et al., Nature (1978)-276, 269-270), FO (deSt. Groth, S. F. and Scheidegger, D., J. Immunol. Methods (1980) 35,1-21), S194 (Trowbridge, I. S., J. Exp. Med. (1978) 148, 313-323), R210(Galfre, G. et al., Nature (1979) 217, 131-133) and the like.

[0077] Cell fusion between the above immune cells and the myeloma cellsmay be essentially conducted in accordance with a known method such asis described in Milstein et al. (Galfre, G. and Milstein, C., MethodsEnzymol. (1981) 73, 3-46) and the like.

[0078] More specifically, the above cell fusion is carried out in theconventional nutrient broth in the presence of, for example, a cellfusion accelerator. As the cell fusion accelerator, for example,polyethylene glycol (PEG), Sendai virus (HVJ) and the like may be used,and an adjuvant such as dimethyl sulfoxide etc. may be added as desiredto enhance efficiency of the fusion.

[0079] The preferred ratio of the immune cells and the myeloma cells foruse is, for example, 1 to 10 times more immune cells than the myelomacells. Examples of culture media to be used for the above cell fusioninclude RPMI 1640 medium and MEM culture medium suitable for the growthof the above myeloma cell lines, and the conventional culture mediumused for this type of cell culture, and besides a serum supplement suchas fetal calf serum (FCS) may be added.

[0080] In cell fusion, predetermined amounts of the above immune cellsand the myeloma cells are mixed well in the above culture liquid, towhich a PEG solution previously heated to about 37° C., for example aPEG solution with a mean molecular weight of 1000 to 6000, is added at aconcentration of 30 to 60% (w/v) and mixed to obtain the desired fusioncells (hybridomas). And then by repeating a sequential addition of asuitable culture liquid and centrifugation to remove the supernatant,cell fusion agents etc. that are undesirable for the growth of thehybridoma can be removed.

[0081] Said hybridoma is selected by culturing in the conventionalselection medium, for example, HAT culture medium (a culture liquidcontaining hypoxanthine, aminopterin, and thymidine). Culturing in saidHAT culture medium is continued generally for the period of timesufficient to effect killing of the cells other than the desiredhybridoma (non-fusion cells), generally several days to several weeks.The conventional limiting dilution method is conducted in which thehybridomas producing the desired antibody are screened and monoclonallycloned.

[0082] In addition to obtaining the above hybridoma by immunizing ananimal other than the human with an antigen, it is also possible toimmunize human lymphocytes in vitro with IL-6 receptor protein or IL-6receptor protein-expressing cells, and the resulting immunizedlymphocytes are fused with a myeloma cell, for example U266, having theability of dividing permanently to obtain a hybridoma that produces thedesired human antibody having the activity of binding to andneutralizing IL-6 receptor (Japanese Post-examined Patent Publication(Kokoku) 1-59878). Furthermore, a transgenic animal having a repertoireof human antibody genes is immunized with the antigen IL-6 receptor orIL-6 receptor-expressing cells to obtain anti-IL-6 receptorantibody-producing cells. The cells are then fused with myeloma cells toobtain hybridomas that are used to obtain human antibody to IL-6receptor (see International Patent Application WO 92-03918, WO 93-12227,WO 94-02602, WO 94-25585, WO 96-33735 and WO 96-34096).

[0083] The monoclonal antibody-producing hybridomas thus constructed canbe maintained in the conventional culture liquid, or can be stored for aprolonged period of time in liquid nitrogen.

[0084] In order to obtain monoclonal antibodies from said hybridoma,there can be mentioned a method in which said hybridoma is cultured inthe conventional method and the antibodies are obtained as thesupernatant, or a method in which the hybridoma is transplanted to andgrown in a mammal compatible with said hybridoma and the antibodies areobtained as the ascites. The former method is suitable for obtaininghighly purified antibodies, whereas the latter is suitable for a largescale production of antibodies.

[0085] In addition to using a hybridoma for antibody production, immunecells such as antibody-producing immunized lymphocytes which has beenimmortalized with an oncogene can be used.

[0086] 3. Recombinant Antibody

[0087] Monoclonal antibodies may be also obtained as a recombinantantibody which has been produced by the recombinant gene technology. Forexample, recombinant antibody can be obtained by cloning a gene of anantibody from a hybridoma or an immune cell such as antibody-producingimmunized lymphocytes, and then integrated into a suitable vector, whichis then introduced into a host to produce said antibody (see, forexample, Borrebaeck, C. A. K. and Larrick, J. W., THERAPEUTIC MONOCLONALANTIBODIES, published in the United Kingdom by MACMILLAN PUBLISHERS LTD.1990).

[0088] Specifically, mRNA encoding the variable region (V region) ofanti-IL-6 receptor antibody is isolated from the hybridoma producinganti-IL-6 receptor antibody. The isolation of mRNA is conducted bypreparing total RNA using, for example, a known method such as theguanidine ultracentrifuge method (Chirgwin, J. M. et al., Biochemistry(1979) 18, 5294-5299), the AGPC method (Chomczynski, P. and Sacchi, N.,Anal. Biochem. (1987) 162, 156-159), and then mRNA is purified from thetotal RNA using the mRNA Purification kit (Pharmacia) and the like.Alternatively, mRNA can be directly prepared using the Quick Prep mRNAPurification Kit (Pharmacia).

[0089] cDNA of the V region of antibody may be synthesized from the mRNAthus obtained using a reverse transcriptase. cDNA may be synthesizedusing the AMV Reverse Transcriptase First-strand cDNA Synthesis Kit(Seikagaku Kogyo), and the like. Alternatively, for the synthesis andamplification of cDNA, the 5′-Ampli FINDER RACE Kit (Clontech) and the5′-RACE method (Frohman, M. A. et al., Proc. Natl. Acad. Sci. U.S.A.(1988) 85, 8998-9002; Belyavsky, A. et al., Nucleic Acids Res. (1989)17, 2919-2932) which employs polymerase chain reaction (PCR) may beused.

[0090] The desired DNA fragment is purified from the PCR productobtained and may be ligated to vector DNA. Moreover, a recombinantvector is constructed therefrom and then is transfected into E. colietc., which is selected to prepare the desired recombinant vector. Thebase sequence of the desired recombinant vector may be confirmed by aknown method such as the dideoxy nucleotide chain termination method.

[0091] Once the DNA encoding the V region of the desired anti-IL-6receptor antibody has been obtained, it may be ligated to DNA encodingthe constant region (C region) of the desired antibody, which is thenintegrated into an expression vector. Alternatively, DNA encoding the Vregion of the antibody may be integrated into an expression vector whichalready contains DNA encoding the C region of the antibody. The C regionof the antibody may be derived from the same animal species as that ofthe V region, or from a different animal species from that of the Vregion.

[0092] In order to produce anti-IL-6 receptor antibody for use in thepresent invention, the antibody gene is integrated into an expressionvector so as to be expressed under the control of an expressionregulatory region, for example an enhancer and/or a promoter.Subsequently, the expression vector is transformed into a host cell andthe antibody is then expressed therein.

[0093] The antibody gene may be expressed by integrating separately DNAencoding a heavy chain (H chain) and a light chain (L chain) of theantibody into an expression vector and co-transforming the host cell, orby integrating DNA encoding an H chain and an L chain into a singleexpression vector and transforming the host cell (International PatentApplication WO 94-11523).

[0094] 4. Altered Antibody

[0095] As recombinant antibodies for use in the present invention,artificially altered recombinant antibodies such as chimeric antibodyand humanized antibody can be used for the purpose of lowering xenogenicantigenicity against humans. Altered antibodies can have the C regionsof human antibody and antibodies such as chimeric antibody or humanizedantibody can be used. These altered antibodies can be produced usingknown methods.

[0096] Chimeric antibody can be obtained by ligating the thus obtainedDNA encoding the V region of antibody other than human antibody to DNAencoding the C region of human antibody, which is then integrated intoan expression vector and introduced into a host for production of theantibody therein (see European Patent Application EP 125023, andInternational Patent Application WO 92-19759,). Using this known method,chimeric antibody useful for the present invention can be obtained.

[0097] Plasmid coding for the V region of the L chain or the V region ofthe H chain of PM-1 antibody has each been designated as pPM-k3 andpPM-h1, respectively, and E. coli having a respective plasmid has beeninternationally deposited under the provisions of the Budapest Treaty asNCIMB40366 and NCIMB40362 on Feb. 11, 1991 with the National Collectionsof Industrial and Marine Bacteria Limited.

[0098] Humanized antibody which is also called reshaped human antibodyhas been made by transplanting the complementarity determining regions(CDRs) of an antibody of a mammal other than the human, for examplemouse antibody, into the CDRs of human antibody. The general recombinantDNA technology for preparation of such antibodies is also known (seeEuropean Patent Application EP 125023 and International PatentApplication WO 92-19759).

[0099] Specifically, a DNA sequence which was designed to ligate theCDRs of mouse antibody with the framework regions (FRs) of humanantibody is synthesized from several divided oligonucleotides havingsections overlapping with one another at the ends thereof, and theoligonucleotides are then synthesized into one integrated DNA. The DNAthus obtained is ligated to a DNA encoding a C region of human antibodyand then is integrated into an expression vector, which is introducedinto a host for antibody production (see European Patent Application EP239400 and International Patent Application WO 92-19759).

[0100] For the FRs of human antibody being ligated to CDRS, the FR thatmake CDR a favorable antigen-binding site is selected. When desired,Amino acids in the FR of antibody V region may be substituted so thatthe CDR of humanized antibody may form an appropriate antigen bindingsite (Sato, K. et al., Cancer Res. (1993) 53, 851-856).

[0101] A preferred embodiment of humanized antibody for use in thepresent invention includes humanized PM-1 antibody (see InternationalPatent Application WO 92-19759). In the humanized PM-1 antibody, CDRs ofthe PM-1 antibody derived from a mouse have been ligated to the FRs ofthe human antibody REI for the L chain, and the FRs of the humanantibody NEW, and part of the amino acid residues of the FR has beensubstituted to obtain antigen-binding activity.

[0102] In order to produce anti-IL-6 receptor antibody for use in thepresent invention, the antibody gene is integrated into an expressionvector so as to be expressed under the control of an expressionregulatory region, for example an enhancer and/or a promoter.Subsequently, the expression vector is transformed into a host cell andthe antibody is then expressed therein.

[0103] The antibody gene may be expressed by integrating separately DNAsencoding a heavy chain (H chain) and a light chain (L chain) of anantibody into an expression vector and co-transforming the host cell, orby integrating a DNA encoding an H chain and an L chain into a singleexpression vector and transforming the host cell (International PatentApplication WO 94-11523).

[0104] Chimeric antibody consists of the V regions of antibody derivedfrom a mammal other than the human and the C regions derived from humanantibody, whereas humanized antibody consists of the CDRs of antibodyderived from a mammal other than the human and the FRs and the C regionsof antibody derived from human antibody. Accordingly, since the aminoacid sequences derived from a mammal other than the human are reduced toa minimum in the above antibodies, antigenicity thereof in the humanbody is reduced so that they are useful as the active ingredient of thetherapeutic agents of the present invention.

[0105] As the C region of human antibody, there can be used, forexample, Cγ1, Cγ2, Cγ3, or Cγ4. The C region of a human antibody mayalso be modified in order to improve the stability of antibody or of theproduction thereof.

[0106] 5. Antibody Fragments and Modified Antibody

[0107] Antibodies for use in the present invention may be fragments ofantibody or modified versions thereof as long as they bind to IL-6receptor and thereby inhibit the binding of IL-6 and IL-6 receptor toblock signal transmission and to inhibit the biological activity ofIL-6. They are antibody fragments or modified antibodies which, whenused in combination with a nitrogen mustard anticancer agent, have ahigher therapeutic effect for myeloma than IL-6 receptor antibody aloneor a nitrogen mustard anticancer agent alone.

[0108] For example, as fragments of antibody, there may be mentionedFab, F(ab′)2, Fv or single-chain Fv (scFv) in which Fv's of H chain andL chain were ligated via a suitable linker. Specifically antibodies aretreated with an enzyme, for example, papain or pepsin, to produceantibody fragments, or genes encoding these antibody fragments areconstructed, and then integrated into an expression vector, which isexpressed in a suitable host cell (see, for example, Co, M. S. et al.,J. Immunol. (1994) 152, 2968-2976; Better, M. and Horwitz, A. H.,Methods Enzymol. (1989) 178, 476-496; Plucktrun, A. and Skerra, A.,Methods Enzympl. (1989) 178, 497-515; Lamoyi, E., Methods Enzymol.(1986.) 121, 652-663; Rousseaux, J. et al., Methods Enzymol. (1986) 121,663-669; Bird, R. E. and Walker, B. W., Trends Biotechnol. (1991) 9,132-137).

[0109] scFv can be obtained by ligating a V region of an H chain and a Vregion of an L chain of an antibody. In the scFv, the V region of Hchainland the V region of L chain are preferably ligated via a linker,preferably a peptide linker (Huston, J. S. et al., Proc. Natl. Acad.Sci. U.S.A. (1988) 85, 5879-5883). The V region of H chain and the Vregion of L chain in the scFv may be derived from any of theabove-mentioned antibodies. As the peptide linker for ligating the Vregions, any single-chain peptide comprising, for example, 12-19 aminoacid residues may be used.

[0110] DNA encoding scFv can be obtained using a DNA encoding an H chainor a V region of an H chain of the above antibody and a DNA encoding anL chain or a V region of an L chain of the above antibody as thetemplate by amplifying the portion of the DNA encoding the desired aminoacid sequence among the above sequences by the PCR technique with theprimer pair specifying the both ends thereof, and by further amplifyingthe combination of DNA encoding the peptide linker portion and theprimer pair which defines that both ends of said DNA be ligated to the Hchain and the L chain, respectively.

[0111] Once DNAs encoding scFv are constructed, an expression vectorcontaining them and a host transformed with said expression vector canbe obtained by the conventional methods, and scFv can be obtained usingthe resultant host by the conventional methods.

[0112] Antibody fragments may be those antibody fragments part of whichsequence has undergone mutation, substitution, deletion, or insertion.These antibody fragments can be produced by obtaining the gene thereofin a similar manner to that mentioned above and by allowing it to beexpressed in a host. “Antibody” as used in the claim of the presentapplication encompasses these antibody fragments.

[0113] As modified antibodies, anti-IL-6 receptor antibody associatedwith various molecules such as polyethylene glycol (PEG) can be used.“Antibody” as used in the claim of the present application encompassesthese modified antibodies. These modified antibodies can be obtained bychemically modifying the antibodies thus obtained. These methods havealready been established in the art.

[0114] 6. Expression and Production of Recombinant Antibody, AlteredAntibody, and Antibody Fragment

[0115] Antibody genes constructed as mentioned above may be expressedand obtained in a known manner. In the case of mammalian cells,expression may be accomplished using an expression vector containing acommonly used useful promoter, an antibody gene to be expressed, and DNAin which the poly A signal has been operably linked at 3′ downstreamthereof. Examples of the promoter/enhancer include human cytomegalovirusimmediate early promoter/enhancer.

[0116] Additionally, as the promoter/enhancer which can be used forexpression of antibody for use in the present invention, there can beused viral promoters/enhancers such as retrovirus, polyoma virus,adenovirus, and simian virus 40 (SV40), and promoters/enhancers derivedfrom mammalian cells such as human elongation factor 1α (HEF1α).

[0117] For example, expression may be readily accomplished by the methodof Mulligan, R. C. et al. (Nature (1979) 277, 108-114) when SV40promoter/enhancer is used, and by the method of Mizushima, S. et al.(Nucleic Acids Res. (1990) 18, 5322) when HEF1α promoter/enhancer isused.

[0118] In the case of E. coli, expression may be conducted by operablylinking a commonly used promoter, a signal sequence for antibodysecretion, and an antibody gene to be expressed, followed by expressionthereof. As the promoter, for example, there can be mentioned laczpromoter and araB promoter. The method of Ward, E. S. et al. (Nature(1989) 341, 544-546; FASEB J. (1992) 6, 2422-2427) may be used when laczpromoter is used, and the method of Better, M. et al. (Science (1988)240, 1041-1043) may be used when araB promoter is used.

[0119] As a signal sequence for antibody secretion, when produced in theperiplasm of E. coli, the pelB signal sequence (Lei, S. P. et al., J.Bacteriol. (1987) 169, 4379-4383) can be used. After separating theantibody produced in the periplasm, the structure of the antibody isappropriately refolded before use (see, for example, InternationalPatent Application WO 96-30394).

[0120] As the origin of replication, there can be used those derivedfrom SV40, polyoma virus, adenovirus, bovine papilloma virus (BPV), andthe like. Furthermore, for gene amplification in the host cell system,expression vectors can include as selection markers the aminoglycosidetransferase (APH) gene, the thymidine kinase (TK) gene, E. coli xanthineguaninephosphoribosyl transferase (Ecogpt) gene, the dihydrofolatereductase (dhfr) gene, and the like.

[0121] For the production of antibody for use in the present invention,any production system can be used, and the production system of antibodypreparation comprises the in vitro or the in vivo production system.

[0122] As the in vitro production system, there can be mentioned aproduction system which employs eukaryotic cells and the productionsystem which employs prokaryotic cells.

[0123] When eukaryotic cells are used, there are the production systemswhich employ animal cells, plant cells, and fungal cells. Known animalcells include (1) mammalian cells such as CHO cells, COS cells, myelomacells, baby hamster kidney (BHK) cells, HeLa cells, and Vero cells, (2)amphibian cells such as Xenopus oocytes, or (3) insect cells such assf9, sf21, and Tn5. Known plant cells include, for example, thosederived from the Nicotiana family, more specifically cells derived fromNicotiana tabacum which is subjected to callus culture. Known fungalcells include (1) yeasts such as the Saccharomyces family, morespecifically Saccharomyces cereviceae, or (2) mold fungi such as thegenus Aspergillus, more specifically Aspergillus niger.

[0124] When prokaryotic cells are used, there are the production systemswhich employ bacterial cells. Known bacterial cells include Escherichiacoli, and Bacillus subtilis.

[0125] By introducing via transformation the gene of the desiredantibody into these cells and culturing the transformed cells in vitro,the antibody can be obtained. Culturing is conducted in the knownmethods. For example, as the culture liquid for mammalian cells, DMEM,MEM, RPMI1640, IMDM and the like can be used, and serum supplements suchas fetal calf serum (FCS) may be used in combination. In addition,antibodies may be produced in vivo by implanting cells into which theantibody gene has been introduced into the peritoneal cavity of ananimal, and the like.

[0126] As in vivo production systems, there can be mentioned those whichemploy animals and those which employ plants. When animals are used,there are the production systems which employ mammals and insects.

[0127] As mammals, goats, pigs, sheep, mice, and cattle can be used(Glaster, V., SPECTRUM Biotechnology Applications, 1993).

[0128] When mammals are used, transgenic animals can be used. Forexample, antibody genes are inserted into the gene encoding proteinwhich is inherently produced in the milk such as goat β casein toprepare fusion genes. DNA fragments containing the fusion gene intowhich the antibody gene has been inserted are injected to a goat embryo,and the embryo is introduced into a female goat. The desired antibody isobtained from the milk produced by the transgenic goat borne to the goatwho received the embryo or offsprings thereof. In order to increase theamount of milk produced containing the desired antibody produced by thetransgenic goat, hormones may be given to the transgenic goat asappropriate. (Ebert, K. M. et al., Bio/Technology (1994) 12, 699-702).

[0129] Also as insects, silkworms can be used. When silkworms are used,baculovirus into which the desired antibody gene has been inserted isinfected to the silkworm, and the desired antibody can be obtained fromthe body fluid of the silkworm (Maeda, S. et al., Nature (1985) 315,592-594).

[0130] Moreover, when plants are used, tabacco, for example, may beused. When tabacco is used, the desired antibody gene is inserted intoan expression vector for plants, for example pMON 530, and then thevector is introduced into a bacterium such as Agrobacterium tumefaciens.The bacterium is then infected to tobacco such as Nicotiana tabacum toobtain the desired antibody from the leaves of the tobacco (Ma, J. K. etal., Eur. J. Immunol. (1994) 24, 131-138).

[0131] An antibody gene is introduced, as mentioned above, into theseanimals or plants, and then the antibody is produced in such animals andplants and is collected therefrom.

[0132] When antibody is produced in in vitro or in vivo productionsystems, as mentioned above, DNAs encoding an H chain and an L chain ofan antibody are separately integrated into expression vectors and thehosts are transformed simultaneously, or a DNA encoding an H chain andan L chain of an antibody is integrated into a single expression vectorand the host is transformed therewith (International Patent ApplicationWO 94-11523).

[0133] 7. Separation and Purification of Antibody

[0134] Antibodies expressed and produced as described above can beseparated from inside or outside of the host cell and then may bepurified to homogeneity. Separation and purification of antibody for usein the present invention may be accomplished by methods of separationand purification conventionally used for proteins without anylimitation.

[0135] For example, separation and purification of antibody may beaccomplished by combining, as appropriate, column chromatography such asaffinity chromatography, filtration, ultracentrifugation, salting-out,dialysis and the like (Antibodies: A Laboratory Manual, Ed Harlow andDavid Lane, Cold Spring Harbor Laboratory, 1988).

[0136] As the column used for affinity chromatography, there can bementioned Protein A column and Protein G column. Examples of the columnemploying Protein A column are Hyper D, POROS, Sepharose F. F.(Pharmacia) and the like.

[0137] Chromatography other than affinity chromatography includes, forexample, ion exchange chromatography, hydrophobic chromatography,gel-filtration, reverse-phase chromatography, absorption chromatographyand the like (Strategies for Protein Purification and Characterization:A Laboratory Course Manual, Ed Daniel R. Marshak et al., Cold SpringHarbor Laboratory Press, 1996). Furthermore, said chromatography may becarried out using a liquid-phase chromatography such as HPLC, FPLC, andthe like.

[0138] 8. Measurement of Antibody Concentration

[0139] The concentration of antibody obtained as above can be determinedby measurement of absorbance or by the enzyme-linked immunosorbent assay(ELISA) and the like. Thus, when absorbance measurement is employed, theantibody obtained is appropriately diluted with PBS and then theabsorbance is measured at 280 nm, followed by calculation using theabsorption coefficient of, though different with species and subclasses,1.4 OD at 1 mg/ml in the case of human antibody.

[0140] When the ELISA method is used, measurement is conducted asfollows. Thus, 100 μl of goat anti-human IgG antibody diluted to 1 μg/mlin 0.1 M bicarbonate buffer, pH 9.6, is added to a 96-well plate (Nunc),and is incubated overnight at 4° C. to immobilize the antibody. Afterblocking, 100 μl each of appropriately diluted antibody of the presentinvention or samples containing the antibody, or 100 μl of human IgG ofa known concentration as the concentration standard is added, andincubated at room temperature for 1 hour.

[0141] After washing, 100 μl of 5000-fold diluted alkalinephosphatase-labeled anti-human IgG antibody is added, and incubated atroom temperature for 1 hour. After washing, the substrate solution isadded and incubated, followed by measurement of absorbance at 405 nmusing the MICROPLATE READER Model 3550 (Bio-Rad) to calculate theconcentration of the desired antibody based on the absorbance of theconcentration standard IgG.

[0142] For determination of antibody concentration, BIAcore (Pharmacia)can be used.

[0143] 9. Confirmation of the Activity of Antibody

[0144] Evaluation of activity of anti-IL-6 receptor antibody of thepresent invention can be conducted using a commonly known method. IL-6is added to a plate in which IL-6 responsive cells such as HN60.BSF2cells were cultured. Then, evaluation is made in the presence ofanti-IL-6 receptor antibody, using the incorporation of ³H labeledthymidine by IL-6 dependent cells as an index.

[0145] Alternatively, ¹²⁵I-labeled IL-6 and anti-IL-6 receptor antibodyare added to a plate in which IL-6 receptor-expressing cells such asU266 have been cultured and then the amount of ¹²⁵I-labeled IL-6 that isbound to the IL-6-expressing cells is determined for evaluation(Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold SpringHarbor Laboratory, 1988).

[0146] As methods for determining the antigen-binding activity ofanti-IL-6 receptor antibody for use in the present invention, there canbe used ELISA, EIA (enzymeimmunoassay), RIA (radioimmunoassay), or thefluorescent antibody method.

[0147] When ELISA is employed, for example, IL-6 receptor is added to a96-well plate onto which antibody against IL-6 receptor has beenimmobilized, and then samples containing the desired anti-IL-6 receptorantibody, for example a culture supernatant of anti-IL-6 receptorantibody-producing cells or purified antibody, are added thereto.Secondary antibody that recognizes the desired anti-IL-6 receptorantibody, labeled with an enzyme such as alkaline phosphatase is added,and the plate is incubated, washed, and then the enzyme substrate suchas p-nitrophenyl phosphate is added thereto. Then the absorbance ismeasured to evaluate the antigen-binding activity. A soluble IL-6receptor may be used as the IL-6 receptor.

[0148] As methods for measuring the inhibition activity of ligandreceptor binding of the anti-IL-6 receptor antibody for use in thepresent invention, the conventional Cell ELISA or the ligand receptorbinding assay can be used.

[0149] In the case of Cell ELISA, for example, cells expressing IL-6receptor are cultured in a 96-well plate and then immobilized withparaformaldehyde etc. Alternatively, membrane fractions of cellsexpressing IL-6 receptor are prepared and a 96-well plate on which IL-6receptors have been immobilized is prepared. To this are added a samplecontaining the desired anti-IL-6 receptor antibody, for example aculture supernatant of anti-IL-6 receptor antibody-producing cells, andpurified antibody, and IL-6 labeled with a radioisotope such as ¹²⁵I,and then the plate is incubated, washed, and radioactivity is measuredto determine the amount of IL-6 bound to the IL-6 receptor and therebyto evaluate the inhibition activity of ligand receptor binding ofanti-IL-6 receptor antibody.

[0150] In the inhibition assay of IL-6 binding to IL-6 receptor on thecells, cells expressing IL-6 receptors are separated by means ofcentrifugation etc. and resuspended to prepare a cell suspension. Asolution of IL-6 labeled with a radioisotope such as ¹²⁵I, or a mixtureof unlabeled IL-6 and labeled IL-6, and a solution containing anti-IL-6receptor antibody whose concentration has been adjusted are added to thecell suspension. After incubating for a certain period of time, thecells are separated, and the radioactivity of the labeled IL-6 bound tothe cell is measured.

[0151] For evaluation of activity of the above antibody, BIAcore(Pharmacia) can be used.

[0152] 10. Method of Administration and Pharmaceutical Preparation

[0153] In accordance with the present invention, a nitrogen mustardanticancer agent. and anti-IL-6 receptor antibody are used incombination. “Used in combination” as used herein refers to a case inwhich pharmaceutical compositions are administered at different times, acase in which pharmaceutical compositions are administered at the sametime, and a case in which one pharmaceutical composition comprising bothof a nitrogen mustard anticancer agent and anti-IL-6 receptor antibodyis administered. In the former two cases, a pharmaceutical compositioncomprising a nitrogen mustard anticancer agent and a pharmaceuticalcomposition comprising anti-IL-6 receptor antibody may be given throughthe same administration route, or a different administration route. Eachof these pharmaceutical compositions is given to cure or inhibit atleast partially the pathological symptoms of patients suffering fromdiseases. The period of administration may be chosen, as appropriate,depending on the age and conditions of the patient.

[0154] Preferably, pharmaceutical compositions comprising anti-IL-6receptor antibody may be administered parenterally, for example viaintravenous injection, drip infusion, intramuscular injection,intraperitoneal injection, subcutaneous injection, and the like, eithersystemically or locally. As local dosage-forms, preferably, externalpreparations, local injections, and the like are used. Externalpreparations are chosen from liniments such as ointments, gel, cream,emulsions, and liquids, tapes, plaster tapes such as patches, or nebulassuch as sprays and powders.

[0155] The effective dosage of anti-IL-6 receptor antibody is chosenfrom the range of 0.001 mg to 1000 mg per kg of body weight per day.Preferably, the dosage is selected from the range of 0.01 to 50 mg perbody weight. The above doses depend on the pathological conditions, andhence they are not limited to these values. The number of administrationis usually selected from, but not limited to, once or twice per day,once per two to a few days, or once per one to four weeks.

[0156] Pharmaceutical compositions comprising a nitrogen mustardanticancer agent are preferably administered orally, but depending onthe nature of the active ingredient, the conditions of patients, and thelike, they may be given parenterally as well. For example intravenousinjection, drip infusion, intraarterial injection, intramuscularinjection, intratumor injection, intrathdracic injection, orintraperitoneal injection, either systemically or locally.

[0157] The effective dosage of nitrogen mustard anticancer agents isdifferent on their kind, but for melphalan, for example, oraladministration of 1 to 20 mg per day, every day or 1 to 6 times perweek, or as high-dose intravenous injection or infusion, single ormultiple doses of 20 to 200 mg/m² is employed. For cyclophosphamide,oral or intravenous administration of 50 to 2000 mg per dose usually forone to 5 times per week, to once per two weeks to one month is employed.The number and the schedule of administration are not limited to thosementioned above. Nitrogen mustard anticancer agents may be given notonly alone but also in combination with vincristine, adriamycin,prednisolone, and the like, as appropriate.

[0158] When a pharmaceutical composition comprising a nitrogen mustardanticancer agent is administered simultaneously with anti-IL-6 receptorantibody, the ratio, is, when combined with daily oral administration ofmelphalan, 0.01 to 1000 fold (weight ratio) relative to the dose ofmelphalan, though it is different on the conditions of the patient andthe administration schedule. Alternatively a pharmaceutical compositioncomprising a certain ratio of the two agents may be administered.However, as mentioned above, the dose ratio varies with the conditionsof the patient etc., and hence it is not limited to the ratio mentionedabove.

[0159] It is also possible to set up a schedule in which apharmaceutical composition comprising a nitrogen mustard anticanceragent and anti-IL-6 receptor antibody are given at different timepoints. For example, to patients for whom remission was introduced byapplying a nitrogen mustard anticancer agent or a combined therapyincluding the agent as a constituent element, anti-IL-6 receptorantibody can be administered in order to maintain remission.Furthermore, administration of a nitrogen mustard anticancer agent or acombined therapy having the agent as a constituent element andadministration of anti-IL-6 receptor antibody may be repeated every 1 to4 weeks. For a nitrogen mustard anticancer agent and anti-IL-6 receptorantibody, preferably the former is given first, but the latter may begiven first depending on the conditions of the patient etc.

[0160] Pharmaceutical compositions of the present invention comprisingnitrogen mustard anticancer agents, pharmaceutical compositionscomprising anti-IL-6 receptor antibody, and pharmaceutical compositionscomprising a nitrogen mustard anticancer agent and anti-IL-6 receptorantibody of the present invention may contain pharmaceuticallyacceptable carriers and/or additives depending on the route ofadministration.

[0161] Examples of such carriers or pharmaceutical additives includewater, a pharmaceutically acceptable organic solvent, collagen,polyvinyl alcohol, polyvinylpyrrolidone, a carboxyvinyl polymer, sodiumcarboxymethylcellulose, sodium, polyacrylate sodium alginate,water-soluble dextran, sodium carboxymethyl starch, pectin, methylcellulose, ethyl cellulose, xanthan gum, gum Arabic, casein, gelatin,agar, diglycerin, glycerin, propylene glycol, polyethylene glycol,Vaseline, paraffin, stearyl alcohol, stearic acid, human serum albumin(HSA), mannitol, sorbitol, lactose, pharmaceutically acceptablesurfactants and the like.

[0162] Actual additives are chosen from, but not limited to, the aboveor combinations thereof depending on the dosage form of a therapeuticagent of the present invention.

[0163] The present invention also encompasses a simultaneous orsequential combined administration of a pharmaceutical agent of thepresent invention with another agent, a biological agent, or a syntheticagent. Other agents are selected from anti-inflammatory agents,antiallergic agents, anti-platelet agents, other anticancer agents orthose that enhance or supplement the activity of the object of thepresent invention.

EXAMPLES

[0164] The present invention will now be explained hereinbelow in moredetail with reference to the following reference examples, experimentalexamples and working examples. It is to be noted that the presentinvention is not limited to these examples in any way.

Reference Example 1. Construction of the Anti-IL-6 Receptor AntibodyPM-1

[0165] The anti-IL-6 receptor antibody MT18 prepared by the method ofHirata et al. (J. Immunol. (1989) 143, 2000-2006) was bound toCNBr-activated Sepharose 4B (manufactured by Pharmacia Fine Chemicals,Piscataway, N.J.) in accordance with the attached directions to purifyIL-6 receptor (Yamasaki et al., Science (1988) 241, 825-828).

[0166] Thus, a human myeloma cell line U266 was solubilized in 1 mMp-paraaminophenylmethane sulphonylfluoride hydrochloride (manufacturedby Wako Chemicals) (digitonin buffer) containing 1% digitonin(manufactured by Wako Chemicals), 10 mM triethanolamine (pH 7.8), and0.15M. NaCl, and then mixed with MT18 antibody conjugated to Sepharose4B beads. The beads were then washed six times with the digitonin bufferto prepare partially purified IL-6 receptor to be used for immunization.

[0167] BALB/c mice were immunized four times every 10 days with theabove-mentioned partially purified IL-6 receptor obtained from 3×10⁹U266 cells, and then hybridomas were prepared in a conventional method.A culture supernatant of hybridomas from growth-positive wells wasevaluated for its ability of binding to IL-6 receptor by the methoddescribed below. 5×10⁷ U266 cells were labeled with ³⁵S-methionine (2.5mCi) and were solubilized in the above digitonin buffer.

[0168] The solubilized U266 cells were mixed with 0.04 ml of MT18antibody conjugated to Sepharose 4B beads and then washed six times inthe digitonin buffer. Using 0.25 ml of the digitonin buffer (pH 3.4),³⁵S-methionine labeled IL-6 receptor was eluted, which was neutralizedwith 0.025 ml of 1M Tris, pH 7.4. The hybridoma culture supernatant 0.05ml was mixed with 0.01 ml Protein G Sepharose (manufactured byPharmacia).

[0169] After washing, the Sepharose was incubated with 0.005 ml solutionof ³⁵S-labeled IL-6 receptor prepared above. Immunoprecipitatingsubstances were analyzed by SDS-PAGE to search the culture supernatantsof hybridoma that reacts with IL-6 receptor. As a result, areaction-positive hybridoma clone PM-1 was established. The anti-IL-6receptor antibody PM-1 produced from the hybridoma PM-1 had the IgG1 κsubtype.

[0170] The activity of the antibody produced by the hybridoma PM-1 toinhibit the binding of IL-6 to IL-6 receptor was evaluated using a humanmyeloma cell line U266. Recombinant human IL-6 was prepared from E. coli(Hirano et al., Immunol. Lett. (1988) 17, 41), and was labeled with ¹²⁵Iusing the Bolton-Hunter reagent (New England Nclear, Boston, Mass.)(Taga et al., J. Exp. Med. (1987) 166, 967).

[0171] 4×10⁵ U266 cells were cultured with a culture supernatant of70%(v/v) hybridoma PM-1 and 14000 CPM of ¹²⁵I-labeled IL-6 at roomtemperature for one hour in the presence of a 100-fold excess ofnon-labeled IL-6. Seventy microliters of a sample was layered onto 300μl of FCS in a 400 μl microfuge polyethylene tube, centrifuged, and thenthe radioactivity of the cells was measured. The result revealed thatthe antibody produced by the hybridoma PM-1 inhibits the binding of IL-6to IL-6 receptor.

Reference Example 2 Construction of a Reshaped Human PM-1 Antibody

[0172] A reshaped human PM-1 antibody was obtained by the methoddescribed in International Patent Application WO 92-19759. From thehybridoma PM-1 prepared in Reference example 1, total RNA was preparedin the conventional method, from which single-stranded cDNA wassynthesized. By the polymerase chain reaction (PCR) method, DNA encodingthe V region of mouse PM-1 antibody was amplified. The primers used inthe PCR method are those described in Jones, S. T. et al.,Bio/Technology (1991) 9, 88-89, 1991.

[0173] The PCR-amplified DNA fragments were purified to obtain DNAfragments containing the gene encoding the V region of mouse kappa-typeL chain and DNA fragments containing the gene encoding the V region ofmouse gamma-type H chain. These DNA fragments were ligated to a plasmidpUC19, which was then transfected into competent E. coli cells CH5α toobtain an E. coli transformant. From the transformant thus obtained, theabove plasmid was obtained, and the base sequence of the V region-codingregion in the plasmid was determined in a conventional method, and thecomplementarity determining region (CDR) of each V region wasidentified.

[0174] In order to construct vectors that express chimera PM-1 antibody,cDNAs encoding the V region of κ L chain and H chain of mouse PM-1 wereseparately inserted into HCMV expression vectors. In order to constructa reshaped human PM-1 antibody, the CDR of V region of mouse PM-1 wasimplanted to human antibody by the CDR grafting method. In order for theCDR of human antibody to form appropriate antigen-binding sites,substitution of amino acids of the framework region (FR) of antibody Vregion was conducted.

[0175] In order to express genes of L chain and H chain of the reshapedhuman PM-1 antibody thus constructed, DNA encoding the L chain or the Hchain was separately inserted into a vector containing the humanelongation factor 1α (HEF-1α) promoter, and a vector expressing the Lchain or the H chain of the reshaped human PM-1 (hPM-1) antibody wasconstructed. By simultaneously inserting these two expression vectorsinto CHO cells, a cell line that produces reshaped human PM-1 antibody(hPM-1) was established. The ability of hPM-1 thus obtained to bind tohuman IL-6 receptor was confirmed by ELISA. Furthermore, hPM-1 inhibitedthe binding of human IL-6 to human IL-6 receptor in a similar manner tothat of mouse antibody and chimeric antibody.

Example 1 The Effects of Combined Use of Anti-human IL-6 ReceptorAntibody and a Chemotherapeutic Agent on the Growth of Human MyelomaCells

[0176] The effects of anti-human IL-6 receptor antibody on sensitivityof KPMM2 cells to chemotherapeutic agents used for treatment of myelomasuch as adriamycin (ADR, manufactured by Kyowa Hakko), vincristine (VCR,manufactured by Sigma Chemical Co.), and melphalan (L-PAM, manufacturedby Sigma Chemical Co.) were evaluated.

[0177] KPMM2 is a multiple myeloma cell line derived from the ascites ofa human patient with myeloma (see Japanese Unexamined Patent Publication(Kokai) No. 7-236475). The patient with myeloma had maintained remissionby the MCNU (ranimustine) and MP (melphalan, prednisolone) therapy, butthe disease recurred and the subsequent VAD (vincristine, adriamycin,dexamethasone) therapy was ineffective. The growth of KPMM2 cells ispromoted by IL-6 and is markedly inhibited by anti-IL-6 antibody oranti-IL-6 receptor antibody (Rinsho Ketueki [The Japanese Journal ofClinical Hematology] (1994) 35, 1361-1365). The growth activity of thecell was evaluated by incorporation of ³H-labeled thymidine(manufactured by Amersham) into the cell.

[0178] The KPMM2 cells that had been maintained were washed thoroughlywith a fresh medium (RPMI1640 medium supplemented with 20% FBS), andthen were adjusted to 4×10⁵/ml, which was dispensed in 50 μl aliquots ina 96-well flat-bottomed microtiter plate (manufactured by Falcon).Furthermore, a medium containing a recombinant human IL-6 (Asagoe, Y. etal., Bio/Technology (1988) 6, 806-809), anti-human IL-6 receptorantibody hPM-1 (see the above reference example 1 or InternationalPatent Application WO 92-15759), and the above chemotherapeutic agentsor a fresh medium as control were added to make 200 μl in each well.

[0179] The plate was incubated at 37° C. for 4 days under the humidifiedcondition in the presence of 5% CO₂. At four hours before the end ofincubation, 10 μl of ³H-labeled thymidine solution (100 μCi/ml) wasadded to each well, and then incubated for more 4 hours. At the end ofincubation, the cells were collected onto the glass filter (manufacturedby Printed Filtermat A, WALLAC) using a harvester (Micro 96 Harvester,manufactured by SKATRON Instruments). The radioactivity incorporatedinto the cells was measured by a microbeta (1450 MicroBeta, manufacturedby WALLAC).

[0180] The activity of growth inhibition on KPMM2 was expressed usingthe effect by a chemotherapeutic agent alone as the control. Using theincorporation of ³H-labeled thymidine into the cell for eachexperimental group in which a each concentration of a chemotherapeuticagent was added as 100, the amount of ³H-labeled thymidine incorporatedinto the cell in an experimental group in which anti-human IL-6 receptorantibody was simultaneously added was compared as an index.

[0181] As a result, comparison of the relationship with theconcentration of a chemotherapeutic agent in the presence of a fixedconcentration of anti-human IL-6 receptor antibody revealed that theindex of adriamycin and vincristine was almost constant regardless oftheir concentration (FIG. 3 to FIG. 6), whereas the index decreased withthe increase in melphalan concentration (FIG. 1 and FIG. 2). In thepresence of 1 ng/ml IL-6, the growth index of 10 μg/ml anti-IL-6receptor antibody alone was 33.9, whereas it dropped to 15.5 in theco-existence of 1 μg/ml melphalan. Similar results were obtained in thepresence of 0.1 μg/ml of IL-6 and the index of the antibody alone was28.4, whereas in the co-existence of 1 μg/ml melphalan, it was 15.9.Thus, combined use of anti-IL-6 receptor antibody and melphalan wasshown to have a synergistic effect.

Example 2 The Effects of Combined Use of Anti-human IL-6 ReceptorAntibody and a Chemotherapeutic Agent in a Human Myeloma Cell-implantedSCID Mouse System

[0182] It was shown in working example 1 that anti-human IL-6 receptorantibody enhances the antitumor effect of chemotherapeutic agents. Amongthem, melphalan (manufactured by Sigma Chemical Co.) which was found toact in a synergistic manner was used to study an in vivo effect ofcombined use.

[0183] For evaluation of antitumor activity, a xenograft model was used.Thus, KPMM2, a human myeloma cell line derived from ascites of a patientwith multiple myeloma was implanted to a male SCID mouse (FOX CHASE C.B17/Icr-Scid Jcl, purchased from Nippon Klea) via the tail vein. At thistime, the tumor cells grow in the bone marrow and come to producemyeloma protein (M protein) in the peripheral blood. Furthermore, thismodel system is very close to an actual clinical condition in that itdevelops major symptoms of multiple myeloma in humans such as bonedisorders, elevated blood calcium, and the like.

[0184] Single cell suspension of myeloma cells for implantation wasprepared by passing through a mesh the well minced KPMM2 cells that havebeen maintained in vivo. Cell density was adjusted to 3×10⁷/ml, whichwas implanted at 0.2 ml per mouse via the tail vein (6×10⁷ cells permouse). The day when the cells were implanted was set at day 0.

[0185] With regard to anti-human IL-6 receptor antibody, a stocksolution that had been preserved at 12.1 mg/ml was diluted in sterilephosphate buffer to make 5 mg/ml. This was given to mice at 0.2 ml peranimal via the tail vein on day 8 (1 mg per mouse). The control groupreceived sterile phosphate buffer containing no antibody in a similarmanner.

[0186] Melphalan (L-PAM, manufactured by Sigma Chemical Co.) that wassuspended at 0.3 or 0.1 mg/ml in 0.2% CMC (carboxymethylcellulose)solution in water was used. This was given orally at 0.1 ml per 10 gbody weight of mouse (3 or 1 mg/kg weight) for 5 consecutive daysstarting on day 1. The control group received 0.2% CMC solution in watercontaining no melphalan in a similar manner.

[0187] The experiment was carried out on the following six groups: (1)the melphalan and antibody non-administration group; (2) the 1 mg/kgmelphalan single-drug administration group; (3) the 3 mg/kg melphalansingle-drug administration group; (4) antibody single-drugadministration group; (5) the 1 mg/kg melphalan and antibody combinedadministration group; and (6) the 3 mg/kg melphalan and antibodycombined administration group. Group 1 included 9 mice, whereas each ofthe other groups included 7 mice. Furthermore, tumor was not implantedon mice of the same lineage and purchased on the same day, which wereused as negative control for M protein detection.

[0188] Indices of the drug efficacy included the survival period, thesurvival ratio without disease on day 120, and the amount of M proteinon day 30. For analysis using a survival curve, a generalized Wilcoxon'stest (SPSS for windows ver. 6, SPSS inc.) was used. A significance levelof 5% or lower was considered to be significant.

[0189] Serum M protein was detected as human IgG using the ELISA method.First, mouse serum was dispensed into a 96-well micro plate that hadpreviously been coated with anti-human IgG antiserum and was allowed tostand. Then alkaline phosphatase-conjugated human IgG antibody was boundthereto, and SIGMA104 phosphatase substrate was added for colordevelopment, absorbance of which was read using a microplate reader. Mprotein content in the serum was calculated using the standard curveobtained from normal human IgG.

[0190] In the anti-IL-6 receptor antibody single-drug administrationgroup or the 1 mg/ml melphalan administration group, no life elongationeffect was observed as compared to the non-administration group. But thecombined use of them significantly elongated life span as compared tothe non-administration group or both of the single-drug administrationgroups (FIG. 7). Furthermore, in the measurement of M protein in theserum on day 30 after implantation, the combined use of them reduced Mprotein level (FIG. 8). In the case of 3 mg/kg melphalan, a significantlife elongation effect was observed for the melphalan single-drugadministration group as compared to the non-administration group, but byusing antibody in combination a significant life elongation effect waseven observed compared to the single-drug administration (FIG. 9). Thesurvival ratio without disease on day 120 was 2/7 in the 3 mg/kgmelphalan single-drug administration group, but the ratio improved to4/7 in the antibody combined administration group (Table 1).

[0191] The administration of melphalan caused toxicity in mice andinhibited body weight gain (FIG. 10). When melphalan was used incombination with anti-IL-6 receptor antibody, an antitumor effect wasenhanced but it did not expand toxicity (inhibition of body weightgain). TABLE 1 Extension of survival period by combined use ofanti-human IL-6 receptor antibody hPM-1 and melphalan Life- None hPM-1Melphalan Survival elongation disease administration dosage n periodratio* (%) ratio — CMC control 9  46.9 ± 1.5 100 0/9 — 1 mg/kg 7  47.3 ±3.1 101 0/7 — 3 mg/kg 7  84.1 ± 9.1 175 2/7 day 8 CMC control 7  55.1 ±5.2 118 0/7 day 8 1 mg/kg 7  68.6 ± 7.5 146 0/7 day 8 3 mg/kg 7 110.0 ±5.1 235 4/7

[0192] It was revealed that by using anti-IL-6 receptor antibody andmelphalan in combination, the life elongation effect was significantlyenhanced in KPMM2 which is a cell line derived from a patient who wasresistant to MP and VAD therapy.

Example 3 The Effects of Combined Use of Anti-human IL-6 ReceptorAntibody and a Chemotherapeutic Agent in a Human Myeloma Cell-implantedSCID Mouse System—a Study on in Vivo Dose Dependency of Antibody

[0193] Example 2 has shown that anti-human IL-6 receptor antibodyenhances the antitumor effect of melphalan. Accordingly, we studied dosedependency of anti-human IL-6 receptor antibody in the combinedadministration of anti-human IL-6 receptor antibody and melphalan.

[0194] For evaluation of antitumor effects, a xenograft model animalthat was produced by implanting KPMM2 cells via the tail vein was usedas in Working example 2. Thus, single-cell suspensions which wereprepared by passing through a mesh KPMM2 cells that were minced afterhaving been maintained in vivo were implanted at 0.2 ml per mouse viathe tail vein (6×10⁶ cells per mouse). The day when the cells wereimplanted was set at day 0.

[0195] With regard to anti-human IL-6 receptor antibody hPM-1, a stocksolution that had been preserved at 6.57 mg/ml was diluted in sodiumphosphate buffer to make each solution of 5, 1, 0.2, and 0.04 mg/ml.They were given to mice at 0.1 ml per 10 g body weight of mouse via thetail vein on day 14 to create the 50, 10, 2, and 0.4 mg/kgadministration groups. The control group received the same bufferwithout antibody in a similar manner.

[0196] Melphalan (L-PAM, manufactured by Sigma Chemical Co.) was used asa suspension of 0.1 mg/ml in 0.2% CMC solution in water. This was givenorally at 0.1 ml per 10 g body weight of mouse (1 mg/kg weight) for 5consecutive days starting on day 7. The control group received 0.2% CMCsolution in water without melphalan in a similar manner.

[0197] The experiment was carried out on the following 10 groups: (A)the melphalan and the antibody non-administration group; (B) themelphalan single-drug administration group; (C) the each dose ofantibody single-drug administration group, 4 groups (50, 10, 2, 0.4mg/kg weight); and, (D) the melphalan and the each dose of antibodycombined administration group, 4 groups (50, 10, 2, 0.4 mg/kg weight).The non-administration group included 12 mice per group, the melphalanadministration group 6 mice per group, whereas each of the other groupsincluded 7 mice. Furthermore, mice of the same lineage and purchased onthe same day were raised without implanting a tumor, and the animalswere used as negative control for M protein detection. The amount of Mprotein was calculated as described in Example 2.

[0198] Indices of the drug efficacy included the survival period, andthe amount of M proteins on day 30, day 35, and day 42. For analysis ofthe survival period, a generalized Wilcoxon's test (SPSS for Windowsver. 6, SPSS inc.) was used. A significance level of 5% or lower wasconsidered to be significant. For analysis of the amount of M protein inthe serum, the ANOVA (Analysis of variance, SPSS for windows ver. 6,.SPSS inc.) was first conducted. After confirming significance,Bonferroni method (SPSS for windows ver. 6, SPSS inc.) was used, and asignificance level of 5% or lower was considered to be significant.

[0199] Since death cases occurred on day 35 in the non-administrationgroup and the each dose of antibody single-drug administration group,they were compared for the amount of M protein on day 30. On the otherhand, in the melphalan single-drug administration group, and themelphalan and antibody combined administration group, the amount of Mprotein on day 30 was very low. Since the anti-human IL-6 receptorantibody that had been administered was detected as M protein thusaffecting the assay, they were compared for the data on day 35 and day42.

[0200] On day 30 in the antibody single-drug administration group, noneof the doses significantly inhibited the amount of M protein, but thesingle-drug administration of melphalan significantly inhibited this(FIG. 11). Then, on day 35 and 42, the effect of combined use ofantibody administration relative to the melphalan administration wasstudied. The result revealed that the combined use of anti-human IL-6receptor antibody at 10 mg/kg, 2 mg/kg, and 0.4 mg/kg significantlyreduced the amount of M protein (FIG. 12, 13).

[0201] For the survival period, none of the doses in the antibodysingle-drug administration group gave a significant life elongationeffect. Furthermore, melphalan alone has shown a significant lifeelongation effect, which was further enhanced by combined use ofantibody (Table 2). At any dose, the life elongation effect tended toincrease. Furthermore, in the 0.4 mg/kg and 50 mg/kg administrationgroups, significance was observed relative to the melphalan single-drugadministration group in the generalized Wilcoxon's test (FIG. 14). TABLE2 Extension of survival period by combined use of anti-human IL-6receptor antibody hPM-1 and melphalan Life- hPM-1 Melphalan Survivalelongation administration dosage n period ratio (%) — CMC control 1241.0 ± 1.7 100  50 mg/kg CMC control 7 41.6 ± 1.0 101  10 mg/kg CMCcontrol 7 40.4 ± 1.7  99   2 mg/kg CMC control 7 41.4 ± 1.3 101 0.4mg/kg CMC control 7 40.3 ± 1.2  98 — 1 mg/kg 6 58.2 ± 1.8 142 (100)  50mg/kg 1 mg/kg 7 65.7 ± 3.9 160 (113)  10 mg/kg 1 mg/kg 7 64.3 ± 2.7 157(111)   2 mg/kg 1 mg/kg 7 63.3 ± 2.3 154 (109) 0.4 mg/kg 1 mg/kg 7 63.7± 1.1 155 (110)

[0202] From the foregoing, it was demonstrated, the combined use ofanti-IL-6 receptor antibody and melphalan has shown an antitumor effectat any dose from 0.4 mg/kg through 50 mg/kg.

[0203] The administration of melphalan caused toxicity to mice andinhibited body weight gain. When melphalan was used in combination withanti-human IL-6 receptor antibody, an antitumor effect was enhanced buttoxicity (inhibition of body weight gain) was not expanded. Therefore,it was suggested that in the treatment of myeloma the administration ofmelphalan may be useful in enhancing effects, reducing the dosage, andbreaking melphalan-resistance.

[0204] Reference to microorganisms deposited under Patent CooperationTReaty Rule 13(2):

[0205] The name and adddress of the depository institute Depositoryorganization: the National Institute of Bioscience and Human Technology,

[0206] Agency of Industrial Science and Technology Address: 1-3, Higashi1-chome, Tsukuba-shi, Ibaraki, Japan

[0207] Accession No. FERM BP-2998

[0208] Date deposited Jul. 10, 1990

[0209] Depository organization: National Collections of Industrial andMarine Bacteria Limited

[0210] Address: 23 St Macher Drive, Aberdeen AB2 IRY, UNITED KINGDOM

[0211] NCIMB 40366 Feb. 11, 1991

[0212] NCIMB 40362 Feb. 11, 1991

What is claimed is:
 1. A method for treating myeloma, comprisingadministering a nitrogen mustard anticancer agent in combination with ananti-IL-6 receptor antibody as part of a treatment regimen, wherein thenitrogen mustard anticancer agent is administered in an amount toenhance the therapeutic effect of the anti-IL-6 receptor antibody. 2.The method according to claim 1, wherein the anti-IL-6 receptor antibodyis a monoclonal antibody.
 3. The method according to claim 2, whereinthe monoclonal antibody is a PM-1 antibody.
 4. The method according toclaim 3, wherein the PM-1 antibody is a reshaped human PM-1 antibody. 5.The method according to claim 1, wherein the nitrogen mustard anticanceragent is mechlorethamine, nitrogen mustard N-oxide, melphalan,uramustin, ifosfamide, chlorambucil, or cyclophosphamide.
 6. The methodaccording to claim 1, wherein the nitrogen mustard anticancer agent ismelphalan and the anti-IL-6 receptor antibody and nitrogen mustard,anticancer agent provide a synergistic effect.
 7. A method for treatingmyeloma, comprising administering an anti-IL-6 receptor antibody incombination with a nitrogen mustard anticancer agent as part of atreatment regimen, wherein the anti-IL-6 receptor antibody isadministered in an amount to enhance the therapeutic effect of thenitrogen mustard anticancer agent.
 8. The method according to claim 7,wherein the anti-IL-6 receptor antibody is a monoclonal antibody.
 9. Themethod according to claim 8, wherein the monoclonal antibody is a PM-1antibody.
 10. The method according to claim 9, wherein the PM-1 antibodyis a reshaped human PM-1 antibody.
 11. The method according to claim 7,wherein the nitrogen mustard anticancer agent is mechlorethamine,nitrogen mustard N-oxide, melphalan, uramustin, ifosfamide,chlorambucil, or cyclophosphamide.
 12. The method according to claim 7,wherein the nitrogen mustard anticancer agent is melphalan and theanti-IL-6 receptor antibody and nitrogen mustard anticancer agentprovide a synergistic effect.